#setwd('/afs/inf.ed.ac.uk/user/s17/s1725186/Documents/PhD-InitialExperiments/Gupta/R_Markdowns')

library(tidyverse) ; library(reshape2) ; library(glue) ; library(plotly) ; library(plotlyutils)
library(RColorBrewer) ; library(viridis) ; require(gridExtra) ; library(GGally)
library(Rtsne)
library(ClusterR)
library(DESeq2)

Load preprocessed dataset (preprocessing code in 19_12_24_data_preprocessing.Rmd)

# Gandal dataset
load('./../Data/preprocessed_data.RData')
datExpr = datExpr %>% data.frame
DE_info = DE_info %>% data.frame

# GO Neuronal annotations
GO_annotations = read.csv('./../../FirstYearReview/Data/GO_annotations/genes_GO_annotations.csv')
GO_neuronal = GO_annotations %>% filter(grepl('neuron', go_term)) %>% 
              mutate('ID'=as.character(ensembl_gene_id)) %>% 
              dplyr::select(-ensembl_gene_id) %>% distinct(ID) %>%
              mutate('Neuronal'=1)

# SFARI Genes
SFARI_genes = read_csv('./../../../Models/FirstPUModel/Data/SFARI/SFARI_genes_08-29-2019_with_ensembl_IDs.csv')
SFARI_genes = SFARI_genes[!duplicated(SFARI_genes$ID) & !is.na(SFARI_genes$ID),]

# Update DE_info with SFARI and Neuronal information
DE_info = DE_info %>% mutate('ID'=rownames(.)) %>% left_join(SFARI_genes, by='ID') %>% 
  mutate(`gene-score`=ifelse(is.na(`gene-score`), 'None', `gene-score`)) %>%
  distinct(ID, .keep_all = TRUE) %>% left_join(GO_neuronal, by='ID') %>%
  mutate(Neuronal=ifelse(is.na(Neuronal), 0, Neuronal)) %>%
  mutate(gene.score=ifelse(`gene-score`=='None' & Neuronal==1, 'Neuronal', `gene-score`), 
         significant=padj<0.05 & !is.na(padj))


SFARI_colour_hue = function(r) {
  pal = c('#FF7631','#FFB100','#E8E328','#8CC83F','#62CCA6','#59B9C9','#b3b3b3','#808080','gray','#d9d9d9')[r]
}

SFARI Gene list

print(paste0('There are ', length(unique(SFARI_genes$`gene-symbol`)), ' genes with a SFARI score'))
## [1] "There are 979 genes with a SFARI score"

There are 979 genes with a SFARI score. but to map them to gene expression mapa we had to map the gene names to their corresponding ensembl IDs

Mapping SFARI Gene names to Ensembl IDs

print(paste0('There are ', nrow(SFARI_genes), ' Ensembl IDs in the SFARI Gene dataset'))
## [1] "There are 1090 Ensembl IDs in the SFARI Gene dataset"



  • Since a gene can have more than one ensembl ID, there were some one-to-many mappings between a gene name and ensembl IDs, so thath’s why we ended up with 1090 rows in the SFARI_genes dataset.

  • 32 genes had their IDs in LRG_n format, so they were manually converted to the regular ENSG…n format except for one that didn’t have any ensembl ID assigned to it (LRG_16, ADA Gene). I assigned it the Ensembl ID associated to the gene directly (ignoring the LRG ID)

  • 15 genes weren’t assigned any ID by bioMart (BICDL1, EMSY, ERBIN, KMT5B, LNPK, MIR137, MSNP1AS, NEXMIF, NSMCE3, PATJ, PLPPR4, PRKN, RP11-1407O15.2, TERB2, TSPOAP1), so they were assigned ensembl IDs manually except for one that didn’t have any ensembl ID assigned to it (MSNP1AS, which is the MSNP1 antisense gene)

  • In the end, 979 SFARI genes could be mapped to an ensembl ID (all except MSNP1AS)

Note: There are 24 genes in the SFARI Gene score list that do not have any score assigned to them, and three of them don’t have a syndromic tag either. I’m not sure what’s going on with these genes or why they are on the list?

cat(paste0('There are ', sum(is.na(SFARI_genes$`gene-score`)) ,
             ' genes in the SFARI list without a score, of which ',
             sum(is.na(SFARI_genes$`gene-score`) & SFARI_genes$syndromic==0),
             ' don\'t have syndromic tag either (Why include them then?????)'))
## There are 24 genes in the SFARI list without a score, of which 3 don't have syndromic tag either (Why include them then?????)

Exploratory Analysis

cat(paste0('There are ', sum(SFARI_genes$ID %in% rownames(datExpr)), ' SFARI Genes in the expression dataset (~',round(100*sum(SFARI_genes$ID %in% rownames(datExpr))/979) ,'%)\n\n'))
## There are 854 SFARI Genes in the expression dataset (~87%)
cat(paste0('Of these, only ', sum(DE_info$`gene-score`!='None'), ' have an assigned score'))
## Of these, only 832 have an assigned score

From now on, we’re only going to focus on these genes with a score

Gene count by SFARI score:

table(DE_info$`gene-score`)
## 
##     1     2     3     4     5     6  None 
##    24    64   180   394   151    19 13427

Gene count by Syndromic tag:

table(DE_info$syndromic)
## 
##   0   1 
## 747 107


GO Neuronal annotations:

cat(glue(sum(GO_neuronal$ID %in% rownames(datExpr)), ' genes have neuronal-related annotations\n\n\n'))
## 1023 genes have neuronal-related annotations
cat(glue('Only ',sum(SFARI_genes$ID %in% GO_neuronal$ID),' of these genes have a SFARI score\n\n'))
## Only 193 of these genes have a SFARI score
table(DE_info$Neuronal[DE_info$`gene-score` %in% c('1','2','3','4','5','6')],
      DE_info$gene.score[DE_info$`gene-score` %in% c('1','2','3','4','5','6')])
##    
##       1   2   3   4   5   6
##   0  16  47 139 325 115  16
##   1   8  17  41  69  36   3


Gene Expression


Normalised data

The higher the SFARI score, the higher the mean expression of the gene but the lower the standard deviation. This is weird because the data was originally heteroscedastic with a positive relation between mean and variance, so the fact that the relation now seems to have reversed could mean that the vst normalisation ended up affecting the highly expressed genes more than it should have when trying to correct their higher variance. The same thing happened with the Gandal dataset.

plot_data = data.frame('ID'=rownames(datExpr), 'MeanExpr'=rowMeans(datExpr), 'SDExpr'=apply(datExpr,1,sd)) %>% 
            left_join(DE_info, by='ID')

p1 = ggplotly(plot_data %>% ggplot(aes(gene.score, MeanExpr, fill=gene.score)) + geom_boxplot() + 
              scale_fill_manual(values=SFARI_colour_hue(r=c(1:6,8,7))) + theme_minimal() +
              theme(legend.position='none'))

p2 = ggplotly(plot_data %>% ggplot(aes(gene.score, SDExpr, fill=gene.score)) + geom_boxplot() + 
              scale_fill_manual(values=SFARI_colour_hue(r=c(1:6,8,7))) + theme_minimal() +
              ggtitle('Mean Expression (left) and SD (right) by SFARI score') + 
              theme(legend.position='none'))

subplot(p1, p2, nrows=1)
rm(plot_data, p1, p2)


Raw data

Just to corroborate that the relation between sd and SFARI score used to be in the opposite direction before the normalisation: The higher the SFARI score, the higher the mean expression and the higher the standard deviation

# Save preprocessed results
datExpr_prep = datExpr
datMeta_prep = datMeta
DE_info_prep = DE_info

load('./../Data/filtered_raw_data.RData')

plot_data = data.frame('ID'=rownames(datExpr), 'MeanExpr'=rowMeans(datExpr), 'SDExpr'=apply(datExpr,1,sd)) %>% 
            left_join(DE_info, by='ID')

p1 = ggplotly(plot_data %>% ggplot(aes(gene.score, MeanExpr, fill=gene.score)) + geom_boxplot() + 
              scale_fill_manual(values=SFARI_colour_hue(r=c(1:6,8,7))) + theme_minimal() +
              theme(legend.position='none'))

p2 = ggplotly(plot_data %>% ggplot(aes(gene.score, SDExpr, fill=gene.score)) + geom_boxplot() + 
              scale_fill_manual(values=SFARI_colour_hue(r=c(1:6,8,7))) + theme_minimal() +
              ggtitle('Mean Expression (left) and SD (right) by SFARI score') + 
              theme(legend.position='none'))

subplot(p1, p2, nrows=1)
rm(plot_data, p1, p2)

Return to normalised version of the data

# Save preprocessed results
datExpr = datExpr_prep
datMeta = datMeta_prep
DE_info = DE_info_prep

rm(datExpr_prep, datMeta_prep, DE_info_prep)


Log Fold Change

It seems there’s a negative relation between SFARI score and log fold change, (although not as strong as with the Gandal dataset) when it would be expected to be either positively correlated, or independent from each other (because there are other factors that determine if a gene is releated to Autism apart from differences in gene expression)

Wikipedia mentions the likely explanation for this: “A disadvantage and serious risk of using fold change in this setting is that it is biased and may misclassify differentially expressed genes with large differences (B − A) but small ratios (B/A), leading to poor identification of changes at high expression levels”.

Based on this, since we saw there is a strong relation between SFARI score and mean expression, assuming the SFARI scores are reliable, log fold change should not be used as a metric to determine differential expression when studying autism.

On top of this, I believe this effect is made more extreme by the pattern found in the previous plots, since the higher expressed genes were the most affected by the normalisation transformation, ending up with a smaller variance than the rest of the data, which is related to smaller ratios. (This is a constant problem independently of the normalisation function used).

ggplotly(DE_info %>% ggplot(aes(x=gene.score, y=abs(log2FoldChange), fill=gene.score)) + 
         geom_boxplot() + scale_fill_manual(values=SFARI_colour_hue(r=c(1:6,8,7))) + 
         theme_minimal() + theme(legend.position='none'))


Effects of modifying filtering threshold by SFARI score

The higher the percentage of genes that get filtered by differential expression. This pattern is not as clear as with Gandal’s dataset

lfc_list = seq(1, 1.15, 0.005)

all_counts = data.frame('group'='All', 'n'=as.character(nrow(DE_info)))
Neuronal_counts = data.frame('group'='Neuronal', n=as.character(sum(DE_info$Neuronal)))
lfc_counts_all = DE_info %>% group_by(`gene-score`) %>% tally %>%
                 mutate('group'=as.factor(`gene-score`), 'n'=as.character(n)) %>%
                 dplyr::select(group, n) %>%
                 bind_rows(Neuronal_counts, all_counts) %>%
                 mutate('lfc'=-1) %>%  dplyr::select(lfc, group, n)

for(lfc in lfc_list){
  
  # Recalculate DE_info with the new threshold (p-values change)
  DE_genes = results(dds, lfcThreshold=log2(lfc), altHypothesis='greaterAbs') %>% data.frame
  
  DE_genes = DE_genes %>% mutate('ID'=rownames(.)) %>% left_join(SFARI_genes, by='ID') %>% 
             mutate(`gene-score`=ifelse(is.na(`gene-score`), 'None', `gene-score`)) %>%
             distinct(ID, .keep_all = TRUE) %>% left_join(GO_neuronal, by='ID') %>%
             mutate(Neuronal=ifelse(is.na(Neuronal), 0, Neuronal)) %>%
             mutate(gene.score=ifelse(`gene-score`=='None' & Neuronal==1, 'Neuronal', `gene-score`))
  
  DE_genes = DE_genes %>% filter(padj<0.05 & abs(log2FoldChange)>log2(lfc))

  
  # Calculate counts by groups
  all_counts = data.frame('group'='All', 'n'=as.character(nrow(DE_genes)))
  Neuronal_counts = data.frame('group'='Neuronal', n=as.character(sum(DE_genes$Neuronal)))
  lfc_counts = DE_genes %>% group_by(`gene-score`) %>% tally %>%
               mutate('group'=`gene-score`, 'n'=as.character(n)) %>%
               bind_rows(Neuronal_counts, all_counts) %>%
               mutate('lfc'=lfc) %>% dplyr::select(lfc, group, n)
  
  
  # Update lfc_counts_all
  lfc_counts_all = lfc_counts_all %>% bind_rows(lfc_counts)
}

# Add missing entries with 0s
lfc_counts_all = expand.grid('group'=unique(lfc_counts_all$group), 'lfc'=unique(lfc_counts_all$lfc)) %>% 
  left_join(lfc_counts_all, by=c('group','lfc')) %>% replace(is.na(.), 0)

# Calculate percentage of each group remaining
tot_counts = DE_info %>% group_by(`gene-score`) %>% tally() %>% filter(`gene-score`!='None') %>%
             mutate('group'=`gene-score`, 'tot'=n) %>% dplyr::select(group, tot) %>%
             bind_rows(data.frame('group'='Neuronal', 'tot'=sum(DE_info$Neuronal)),
                       data.frame('group'='All', 'tot'=nrow(DE_info)))

lfc_counts_all = lfc_counts_all %>% filter(lfc!=-1, group!='None') %>% 
                 left_join(tot_counts, by='group') %>% mutate('perc'=round(100*as.numeric(n)/tot,2))


# Plot change of number of genes
ggplotly(lfc_counts_all %>% ggplot(aes(lfc, perc, color=group)) + geom_point(aes(id=n)) + geom_line() + 
         scale_color_manual(values=SFARI_colour_hue(r=1:8)) + ylab('% of remaining genes') +  xlab('Fold Change') + 
         ggtitle('Effect of filtering thresholds by SFARI score') + theme_minimal())
rm(lfc_list, all_counts, Neuronal_counts, lfc_counts_all, lfc, lfc_counts, lfc_counts_all, tot_counts, lfc_counts_all)

Session info

sessionInfo()
## R version 3.6.2 (2019-12-12)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.3 LTS
## 
## Matrix products: default
## BLAS:   /usr/lib/x86_64-linux-gnu/openblas/libblas.so.3
## LAPACK: /usr/lib/x86_64-linux-gnu/libopenblasp-r0.2.20.so
## 
## locale:
##  [1] LC_CTYPE=en_GB.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_GB.UTF-8        LC_COLLATE=en_GB.UTF-8    
##  [5] LC_MONETARY=en_GB.UTF-8    LC_MESSAGES=en_GB.UTF-8   
##  [7] LC_PAPER=en_GB.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=en_GB.UTF-8 LC_IDENTIFICATION=C       
## 
## attached base packages:
## [1] parallel  stats4    stats     graphics  grDevices utils     datasets 
## [8] methods   base     
## 
## other attached packages:
##  [1] DESeq2_1.24.0               SummarizedExperiment_1.14.1
##  [3] DelayedArray_0.10.0         BiocParallel_1.18.1        
##  [5] matrixStats_0.54.0          Biobase_2.44.0             
##  [7] GenomicRanges_1.36.0        GenomeInfoDb_1.20.0        
##  [9] IRanges_2.18.2              S4Vectors_0.22.0           
## [11] BiocGenerics_0.30.0         ClusterR_1.2.1             
## [13] gtools_3.8.1                Rtsne_0.15                 
## [15] GGally_1.4.0                gridExtra_2.3              
## [17] viridis_0.5.1               viridisLite_0.3.0          
## [19] RColorBrewer_1.1-2          plotlyutils_0.0.0.9000     
## [21] plotly_4.9.0                glue_1.3.1                 
## [23] reshape2_1.4.3              forcats_0.4.0              
## [25] stringr_1.4.0               dplyr_0.8.2                
## [27] purrr_0.3.3                 readr_1.3.1                
## [29] tidyr_0.8.3                 tibble_2.1.3               
## [31] ggplot2_3.2.0               tidyverse_1.2.1            
## 
## loaded via a namespace (and not attached):
##  [1] colorspace_1.4-1       htmlTable_1.13.1       XVector_0.24.0        
##  [4] base64enc_0.1-3        rstudioapi_0.10        bit64_0.9-7           
##  [7] AnnotationDbi_1.46.1   lubridate_1.7.4        xml2_1.2.2            
## [10] splines_3.6.2          geneplotter_1.62.0     knitr_1.23            
## [13] zeallot_0.1.0          Formula_1.2-3          jsonlite_1.6          
## [16] broom_0.5.2            annotate_1.62.0        cluster_2.1.0         
## [19] shiny_1.3.2            compiler_3.6.2         httr_1.4.0            
## [22] backports_1.1.4        assertthat_0.2.1       Matrix_1.2-18         
## [25] lazyeval_0.2.2         cli_1.1.0              later_0.8.0           
## [28] acepack_1.4.1          htmltools_0.3.6        tools_3.6.2           
## [31] gmp_0.5-13.5           gtable_0.3.0           GenomeInfoDbData_1.2.1
## [34] Rcpp_1.0.1             cellranger_1.1.0       vctrs_0.2.0           
## [37] nlme_3.1-143           crosstalk_1.0.0        xfun_0.8              
## [40] rvest_0.3.4            mime_0.7               XML_3.98-1.20         
## [43] zlibbioc_1.30.0        scales_1.0.0           promises_1.0.1        
## [46] hms_0.5.1              yaml_2.2.0             memoise_1.1.0         
## [49] rpart_4.1-15           reshape_0.8.8          latticeExtra_0.6-28   
## [52] stringi_1.4.3          RSQLite_2.1.2          genefilter_1.66.0     
## [55] checkmate_1.9.4        rlang_0.4.2            pkgconfig_2.0.2       
## [58] bitops_1.0-6           evaluate_0.14          lattice_0.20-38       
## [61] labeling_0.3           htmlwidgets_1.3        bit_1.1-14            
## [64] tidyselect_0.2.5       plyr_1.8.4             magrittr_1.5          
## [67] R6_2.4.0               generics_0.0.2         Hmisc_4.2-0           
## [70] DBI_1.0.0              pillar_1.4.2           haven_2.1.1           
## [73] foreign_0.8-73         withr_2.1.2            survival_3.1-8        
## [76] RCurl_1.95-4.12        nnet_7.3-12            modelr_0.1.5          
## [79] crayon_1.3.4           rmarkdown_1.13         locfit_1.5-9.1        
## [82] grid_3.6.2             readxl_1.3.1           data.table_1.12.2     
## [85] blob_1.2.0             digest_0.6.19          xtable_1.8-4          
## [88] httpuv_1.5.1           munsell_0.5.0